Are the Myeloma Bone Microevironment Cells Tumoral or Not?.

Abstract 1816

Poster Board I-842

Multiple myeloma (MM) is characterized by a tight relationship between tumor and bone microenvironment cells that supports the survival of MM cells and determines a severe impairment of bone homeostasis. Mesenchymal/osteoblastic cells have a critical pathophysiological role in this process either through the overproduction of growth factors and osteoclastogenic molecules or undergoing them same to alterations responsible of bone formation suppression that rarely repairs even after disease remission. These evidences suggest that bone microenvironments cells could be directly involved in myelomagenesis although it is still unclear whether bone microenvironment cells are primary tumoral in MM patients.

To clarify this issue, we analyzed immunophenotypic, functional, transcriptional and genomic profiles of bone-derived mesenchymal (MSC) and osteoblastic (OB) cells obtained from a cohort of 24 MM patients at the diagnosis, 7 MGUS patients and 8 healthy donors (N) underwent to orthopedic surgery. MSC cells were isolated from bone biopsies collected in V-glass tube and minced extensively with surgical scissors. OB cells were directly isolated from the minced bone chips collected at the bottom of V-glass tube. The presence of potential haemopoietic and MM contaminating cells was excluded by FACS analysis in both MCSs and OBs, testing CD3, CD14, CD20 and CD138 antigens as well as the expression of CD105, CD146 and the osteoblast markers osteocalcin, alkaline phosphatase, collagen I and Runx2 evaluated in OBs as compared to MSCs Any significant difference in the immunophenotype of MSCs and OBs was not found between MM, MGUS and N with the exception of Runx2 expression that was found to be lower in MM vs. N-MSCs. Cell proliferation and cell doubling/day were found to be higher in MSCs as compared to OBs even if any significant difference was not found across the three groups analyzed. The transcriptional analysis of isolated MSCs and OBs was performed using high-density GeneChip® oligonucleotide microarrays. A multiclass analysis identified 43 differentially expressed genes, which characterized N-MSCs versus MM-MSCs cases but did not distinguish MGUS and MM samples. The molecular signature showed a modulation of genes involved in cell adhesion, cell cycle, transcriptional regulation (MAF and HOX genes) and cell proliferation. A similar transcriptional pattern (78 differentially expressed genes) was observed in N-OBs versus MM-OBs samples. Interestingly, HOXB genes (HOXB2, HOXB6 and HOXB7) were found to be up-regulated in MM as compared to N OBs. Gene expression profiling data were then confirmed by real time PCR. The genomic profiles of MSCs and OBs, obtained from 10 MM patients, were generated by whole-genome array-comparative genomic hybridization (array-CGH) using the 105K platform with a resolution of about 60 kb. Experiments were performed according to the Agilent's protocol v 5.0. DNA either from CD3⁺ or PBC cells or saliva of every patient was used as control DNA in all experiments. No chromosomal abnormality was observed in MSCs whereas the presence of chromosomal rearrangements and trisomy were found in the OBs of about 50% of MM patients. These abnormalities were different to those observed in CD138⁺ MM cells of the same patients and the percentage of OBs carrying these abnormalities increased in the late culture passages. Finally, the presence of the two known telomere maintenance mechanisms, telomerase activity (TA) and alternative lengthening of telomere (ALT) have been investigated in cultures at early passages of MSCs and OBs. TA was determined using the telomeric repeat amplification protocol, a PCR-based method in which telomerase extends a radiolabeled synthetic primer resembling telomeric DNA, in both fresh and frozen MSC and OB cultures obtained from 9 MM and 4 MGUS patients. ALT was detected by assaying ALT-associated promyelocytic leukaemia (PML) nuclear bodies [APB], using a combined technique of PML immunofluorescence and telomere FISH, in fresh MSC and OB cultures obtained from both MM and MGUS patients. Notably, no evidence of TA and ALT expression was found in all the tested cultures of both MSCs and OBs indicating that both cells do not display malignant transformation.

Overall, our preliminary data indicate that in MM patients both MSCs and OBs show transcriptional alterations as compare to controls and OBs may share genomic abnormalities although these are unlike to be correlated with a primary tumoral feature.